Retinal diseases can lead to a devastating loss of vision and have grave personal and societal consequences. They are often modeled in animals, and more recently in human embryonic stem cell (hESC)-derived retina, in efforts to improve understanding of disease pathogenesis and to develop novel therapies. However, human retinal development differs from that of animal and hESC-derived retina models in important yet poorly understood ways. Defining such differences could reveal previously unrecognized human-specific features, identify developmentally important factors in the retina's in vivo ocular environment, and enable more accurate modeling of retinal diseases both in animals and in the hESC-derived retina system. This study aims to improve understanding of human retina features that fail to be modeled in mice or in hESC- derived retina, while also defining the differentiation states of a human retinal cell type in unprecedented detail. The study focuses on the post-mitotic differentiation of human cone photoreceptor precursors. These cells may be especially poorly represented in model systems because they form specialized structures (the macula and fovea) and express a proliferation-related program that is not evident in mouse or in hESC-derived models. We propose to define the differentiation states through which post-mitotic cone precursor's progress in vivo, in developing human and mouse retina, and also in vitro, in hESC-derived retina, and to explore the basis of major discrepant features of the model systems. In each context, we will a) isolate and define transcriptomes of individual cone precursors over a range of differentiation states, b) define and temporally order cone precursor differentiation states, and c) identify dynamically regulated genes and signaling pathways that mark the progression from one state to another. We will then probe the basis of human cone precursor cell state transitions by defining open chromatin regions and conserved cis-regulatory elements in dynamically regulated genes. We will also define the orthologous differentiation states through which human, mouse, and hESC-derived cone precursors progress and the major differences between the three settings, including but not limited to the human cone precursor proliferation-related program. Together, the studies will provide a detailed portrait of human cone precursor differentiation, enable development of more accurate in vivo and in vitro retinal disease models, and provide a novel approach to the study of human retinal development and disease.